The invention concerns a time-of-flight mass spectrometer for the analysis of a large number of samples on a sample carrier using laser desorption and associated analytical procedures.
Time-of-flight mass spectrometers in which the samples are ionized by pulsed laser desorption operate as follows: the laser beam is focused onto a sample that is held on a sample carrier. A laser desorption pulse generates ions from the analyte molecules. An applied voltage accelerates the ions into a field-free flight tube. Because of their different masses, the ions in the ion source are accelerated to different velocities. The smaller ions reach the time-resolving ion detector earlier than the larger ions. From the measured time-resolved ion currents the flight times of the ions can be determined; from their flight times their masses are calculated. An energy-focusing reflector and a delay in acceleration (often called delayed extraction) are used to increase resolution.
There is a strong trend in biochemistry towards processing and analyzing very large numbers of samples in parallel. The chip technique has become particularly well known. This technique works with large arrays of samples in a very small space. For certain analytical procedures using such sample arrays, e.g. those using fluorescence spectrometry, devices (xe2x80x9cchip readersxe2x80x9d) are already available which permit parallel analyses of all the samples on the chip at the same time; if not strictly simultaneously, they use a laser scanning procedure to read sequentially in only a few milliseconds per sample. In contrast to this, mass spectrometric analysis at present requires a few seconds for each sample, so that it is currently slower than the chip reader by a factor of about 1,000.
The chip technique is primarily used in genetics, but also starts in protein analysis. In genetics, various types of oligonucleotide are usually bonded to the surfaces in the individual compartments of the array. These oligonucleotides serve as detection sequences for amplified DNA samples; they are often referred to as probes. If the DNA in the sample contains a strand that is complementary to the oligonucleotide, then the DNA will attach itself to the oligonucleotide. This attachment is referred to as xe2x80x9chybridizationxe2x80x9d. The attached strands of DNA can, in this process, bring fluorescent dyes with them, or can alter the fluorescence wavelength of fluorescent dyes that are already located at the bonded oligonucleotides. Chips charged in this way can then be measured in the chip readers mentioned above, and the fluorescence of a compartment indicates the presence of the DNA sequence in question.
Mutations such as, for instance, point mutations can also be examined in this way. Point mutations consist of the modification of a single nucleotide. They are nowadays referred to as SNPs (single nucleotide polymorphisms). Because, however, the hybridization temperature (also known as the melting temperature) for mutation sequences and wild type sequences are only minimally different, detection through simple hybridization is always liable to error, because mutation DNA and wild type DNA undergo mixed hybridization in specific ratios, where the ratio depends very strongly on the temperature. In contrast to this kind of mutation detection through simple hybridization, mass spectrometric mutation detection is very reliable and unambiguous.
Mass spectrometric mutation detection is usually based on enzymatic modification of the probes that have been attached to the chips. The modification is controlled by the hybridized template, so that information about the mutations is transferred from the templates to the probes. One of these enzymatic modifications is known as limited primer extension. Preparation of the samples requires more time than simple hybridization, and the measurement also takes longer, because there is as yet no known method of analyzing a large number of samples simultaneously in the way that can be done in those chip readers. Mass spectrometric measurement of mutations has, however, the advantage of high analytic reliability, something which plays a significant role in diagnostic procedures as well as, for instance, in association with police records.
In protein analysis, antibody proteins can be bonded to the array compartments, and these can capture specific proteins from a sample. Other types of protein-specific affinity bonding can also be used for the protein chip technique. Further detail will not be given here of protein analysis, of which an extraordinarily wide variety exists.
As has already been stated, mass spectrometry offers the advantage of high analytic reliability, but this is countered by the disadvantage of the long time needed for the measurement, being some 1,000 times slower than chip readers. This raises the questions of whether and how mass spectrometry can be accelerated, and in particular of whether spectrometry can be carried out on entire arrays of samples.
There is an odd discrepancy in the mass spectrometric analysis of samples with ionization by laser desorption:
It is, on the one hand, possible to manufacture pulsed lasers with high repetition rates in the region of 10,000 pulses per second; this would permit a mass spectrum to be recorded in only about 100 microseconds, which in turn would mean that 10,000 individual spectra could be measured each second. If 10 individual mass spectra (from 10 laser pulses) are required for a sum spectrum that can be effectively analyzed, the analysis could be complete within one millisecond, which compares favorably with fluorescence chip readers.
On the other hand, however, there is a threshold energy for the generation of ions by laser desorption, below which the laser desorption process does not generate any ions at all. This threshold energy significantly heats the sample. For this reason, a laser frequency greater than about 20 Hertz results in excessive heating and degradation of many samples. Each laser desorption process, moreover, causes the samples to become electrically charged, since only particles of one polarity are withdrawn, while those with the opposite polarity are accelerated back onto the sample. Time is therefore necessary, particularly for MALDI samples with their insulating matrix crystals, so that the charges can disperse. Dissipation of the charges is also in many cases made slower because the chips that are used as a substrate for the samples are made of poorly conducting semiconductor material. Electrical charge causes the quality of the spectra to deteriorate, because the ions generated by the sequence of laser desorptions are given different energies. So if the 10 individual spectra required are to have good quality, at least about a half a second has until now been required. In addition to this, further time is needed to move the new sample into the laser focus, and for the data to be read from the transient recorders. These times are considerably longer than a millisecond; they limit the frequency with which individual samples can be measured, even if the laser pulse frequency could be significantly raised.
In theory, therefore, a minimum period of about one second is required for the analysis of a sample, although in practice the analysis times for a sample are longer still, and still require two to three seconds even in laboratories used to the routine.
Forty years ago, however, there were already developments in mass spectrometry in which a micrometric/analytic representation of a small area was created through ionization by secondary ions (SIMS=secondary ion mass spectrometry). The leader in the field was the French firm Cameca, whose devices were ion-optical imaging magnetic sector-field mass spectrometers. They were used for surface analysis, usually of metal surfaces. Light/dark images were used to determine the presence of specific elements or compounds, and their distribution over the surface.
Time-of-flight mass spectrometers have also already been used for the xe2x80x9cchemicalxe2x80x9d representation of surfaces by laser desorption, although without using imaging optics, but with a raster scan procedure in which the sample is moved through the focal point of the pulsed laser beam and the xe2x80x9cchemical imagexe2x80x9d composed in a complicated manner from many hundreds of individual mass spectra (for example, the xe2x80x9cLammaxe2x80x9d time-of-flight mass spectrometer from Leybold, developed by Kaufmann and Hillenkamp).
From amongst the various methods of ionization by laser desorption, in recent years the MALDI (matrix assisted laser desorption and ionization) process has gained wide acceptance. The MALDI preparation and measurement process consists of first embedding the analyte molecules on a sample carrier in a solid, UV-absorbing matrix, usually an organic acid. The sample carrier is inserted into the ion source of a mass spectrometer. A short laser pulse, about three nanoseconds long, is used to evaporate the matrix into the surrounding vacuum; the analyte molecule is thus brought into the gas phase. Through impacts with matrix ions created at the same time, the analyte molecule is ionized, if primary ionization has not already occurred during the desorption process.
MALDI is particularly suitable for the analysis of peptides and proteins. The analysis of nucleic acid chains is more difficult, and only achieves adequate quality for short chain nucleic acids. The reason for this is that whereas for the ionization of peptides and proteins only a single proton needs to be captured, for nucleic acids, which are a poly-anion with a sugar-phosphate backbone having a multiple negative charge (one negative charge per nucleotide), the process of ionization by the matrix is significantly less efficient. It only functions sufficiently well for very short chains, such as, for instance, for the cleavage products of the extension primers, such as can be created by photo-cleavable linkers.
MALDI ionization creates ions with widely scattered initial velocities, and these can no longer be time-focused, even if an energy focusing reflector is used. The resulting spectra only have moderate resolution. The mass resolution can be improved through a procedure involving acceleration that is only initiated after a delay.
The invention uses a special beam focusing system for the pulsed laser beam in a time-off-light mass spectrometer to generate a firm-positioned pattern of focal points, inserts a matching pattern of samples on a sample carrier into the positions of the focal points, and focuses the ions generated in the laser focal points by an ion-optical imaging system on to one or more ion detectors.
This can involve a pulsed laser beam being split spatially over a number of laser focal points, so that the samples in the focus pattern are each ionized at the same time, but it is also possible for the laser beam to be deflected over time so that all the samples in the fixed-position focus pattern are subjected to pulsed ionization one after another as in a raster scan. Both the raster scan and the spatially split laser beam pulses are repeated at a frequency that is low enough to be compatible with the samples and the quality of the spectra generated, which depends on the dispersal of electrical charges.
The time-of-flight mass spectrometer according to the invention is defined by the characteristic part of claim 1, and includes those parts of the device that facilitate the above procedure, in particular the beam focusing system for generation of the focus pattern and the ion focusing system for projecting the ions from the focal points onto one or more ion detectors.
In what follows, the term xe2x80x9cfocus patternxe2x80x9d will refer not simply to the arrangement of the focal points, but in a more general sense to all the places where ions are generated from the samples that are to be analyzed at the same time. The focus pattern has a fixed relationship to the ion-focusing imaging system, and also to the laser system, while the samples themselves are located on a moveable sample carrier plate. The focus pattern can, for instance, be a rectangular field with 4, 9, 16, 25 or 36 focus locations, or may also be a hexagonal field with, for instance, 7 focus locations.
The term xe2x80x9cfocal pointxe2x80x9d will refer here to a narrowing of the pulsed laser beam created by a lens or a concave mirror, in such a way that the narrowing creates a pre-determined energy density in a predetermined small area. This is not necessarily the same as focusing in the ordinary sense of the word.
The samples are applied in a pattern to the sample carrier, and the pattern spacing corresponds precisely to the spacing of the focus pattern; it is, however, possible for the sample carrier to have many more samples on it than can be included in one focus pattern. This means that the sample pattern on the sample carrier can be a great deal larger in area than the focus pattern; it is even possible, although this would not have much purpose, for the sample pattern to have a smaller spacing than the focus pattern, so that only every second or third sample would be included in the focus pattern. In any case, it is possible at any one time to move a subset of the total pattern of samples on the sample carrier plate into the focus pattern by moving the sample carrier. The sample carrier then remains stationary until the samples in the subset of the pattern that are now located in the focus pattern have all been analyzed. It is one of the advantages of this procedure that movement of the samples for analysis of the samples in the focus pattern, and therefore also movement of the sample carrier, is no longer necessary, and the time required for this is saved.
The phrase xe2x80x9canalysis of a samplexe2x80x9d here refers to the recording of a mass spectrum capable of analysis, and its further processing. Usually, a number of individual spectra are combined to form a single sum spectrum for each sample, and only the sum spectrum is considered to be an analytically evaluable mass spectrum. The sum spectrum has a significantly better signal to noise ratio than the individual spectra, and in many cases it is only this that allows effective evaluation.
The ion focusing system may project the streams of ions, originating from the point where the focused laser pulses fall on the individual samples in the focus pattern, onto a single, common ion detector, or on a number of ion detectors. It is possible for the samples in the focus pattern to be ionized simultaneously by a spatially split laser beam having a number of focal points, or it is possible for the samples to be treated individually by one laser beam being diverted cyclically to a new sample each time, so that in each cycle the individual spectra of all the samples in the focus pattern are recorded once each, and the addition of the individual spectra from, for example, 10 cycles yield the sum spectra for the samples.
This gives us the three arrangements of interest according to the invention:
1) The ions from the various samples in the focus pattern are created together simultaneously by a split laser beam and are measured together by one detector. This case corresponds to the multiplexed analysis of one sample with a number of analytes, but has the advantage of separate preparation of the samples, which is sometimes necessary, and the further advantage that suppression effects (xe2x80x9cquenchingxe2x80x9d) that are often observed (especially in the case of peptides) during ion generation by the laser desorption pulse do not occur. The laser used here must have an increased beam energy in order to supply the necessary threshold energy to all the samples simultaneously.
2) The ions from the various samples in the focus pattern are created together simultaneously by a split laser beam, but are projected onto different detectors separately through appropriate ion focusing methods, as illustrated in FIG. 1. The signals from the detectors must then be sent in parallel to separate signal processors (post-amplifiers and transient recorders). The laser for this purpose must again have an increased beam energy. The number of post-amplifiers and transient recorders used in parallel must correspond to the number of samples in one focus pattern that are to be analyzed.
3) The ions from the various samples in the focus pattern are ionized, in sequence, in a raster scanning process cyclically by means of rapid diversion of the laser beam, and are measured by a single ion detector with only one post-amplifier and only one transient recorder operating with temporal overlap, as illustrated in FIG. 2. The individual spectra from the samples are stored in separate memory regions of the transient recorder, and, if necessary, may be added there to form a sum spectrum. This requires a laser with a normal energy density in the laser beam, but operating at a frequency increased by a factor corresponding to the number of samples. The transient recorder requires an increased memory size so that it can record all the spectra.
Cases 2) and 3) each supply a separate analysis for each sample. If it is not difficult to increase the laser""s beam energy, and if the transient recorders are economically priced, then solution 2) is certainly to be preferred. If, however, it is more economical to increase the laser pulse frequency, and if the transient recorders are relatively expensive, then solution 3) represents the better method. The invention thus allows the price to be optimised. For cases 1) and 3) it is possible not just to use linear mass spectrometers, but also to make use of time-of-flight mass spectrometers with energy-focusing reflectors.